Anal. Chem. 1996, 68, 746-752
Coupling of Reversed-Phase Liquid Column Chromatography and Fourier Transform Infrared Spectrometry Using Postcolumn On-Line Extraction and Solvent Elimination G. W. Somsen,* E. W. J. Hooijschuur, C. Gooijer, U. A. Th. Brinkman, and N. H. Velthorst
Department of General and Analytical Chemistry, Free University, De Boelelaan 1083, 1081 HV Amsterdam, the Netherlands T. Visser
Laboratory for Organic-analytical Chemistry, National Institute of Public Health and Environmental Protection (RIVM), P.O. Box 1, 3720 BA Bilthoven, the Netherlands
An on-line postcolumn extraction module was used in conjunction with a solvent elimination interface for the semi-on-line coupling of reversed-phase liquid chromatography (RP-LC) and Fourier transform infrared spectrometry (FT-IR). The extraction module consisted of a phase segmentor, an extraction coil, and a phase separator. Dichloromethane was used as extraction solvent. The organic phase delivered by the separator was evaporated by a spray-jet assembly that simultaneously deposited the extracted analytes onto a zinc selenide window, which was subsequently analyzed by FT-IR microscopy. The method is evaluated by studying parameters such as postcolumn band broadening, phase separation efficiency, evaporation efficiency, extraction yield, eluent composition, and use of nonvolatile buffer salts. Good-quality spectra were obtained for test compounds (phenylureas and quinones), which were separated by RP-LC using a buffered eluent with high water content. Large-volume injections allowed FT-IR detection at the submicrogram per milliliter level. Over the years, various techniques that combine column liquid chromatography (LC) and Fourier transform infrared spectrometry (FT-IR) have been developed to provide structural information about components of complex mixtures. In general, two LCFT-IR interfacing methods can be discerned, viz., techniques that use flow cells and techniques involving solvent elimination.1,2 Flow cell LC-FT-IR is hindered by eluent interferences, which usually result in poor sensitivity and reduction of the obtainable spectral information, especially when aqueous LC eluents are used. More successful coupling of LC and FT-IR is accomplished by solvent elimination prior to IR detection. This involves an interface that evaporates the eluent and deposits the analytes onto a medium compatible with FT-IR detection. Using this method, solventabsorption-free FT-IR spectra of analytes can be obtained in the low nanogram range.3-10 However, when using aqueous eluents, (1) Patonay, G., Ed. HPLC DetectionsNewer Methods; VCH: New York, 1992; Chapter 7. (2) Fujimoto, C.; Jinno, K. Anal. Chem. 1992, 64, 476A. (3) Gagel, J. J.; Biemann, K. Anal. Chem. 1987, 59, 1266. (4) Conroy, C. M.; Griffiths, P. R.; Jinno, K. Anal. Chem. 1985, 57, 822.
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as is common in reversed-phase (RP) LC, the high heat of evaporation of water hampers rapid eluent elimination. Therefore, in RP-LC-FT-IR, sophisticated interfaces with an enhanced solvent evaporation power are required. Furthermore, reduction of the chromatographic flow rate is often necessary in order to minimize the eluent volume that has to be eliminated. In the literature, several solvent elimination interfaces for RPLC-FT-IR are described. Gagel and Biemann3 used an interface design in which solvent evaporation is facilitated by mixing the aqueous column effluent with nitrogen gas. To increase the evaporation rate of aqueous eluents, Lange and co-workers6 used a concentric flow nebulizer housed in a vacuum chamber. Raynor et al.8 studied the feasibility of electrospray nebulization for RPLC-FT-IR. The particle beam interface, originally developed for LC-mass spectrometry, was modified for LC-FT-IR by de Haseth and co-workers.11-13 Robertson et al.14,15 used a thermospray interface to combine RP-LC and FT-IR. All RP-LC-FT-IR systems mentioned above use direct elimination of the aqueous eluent. This frequently implies that one has to compromise with regard to eluent water content, eluent flow rate, or IR sensitivity. The tedious evaporation of water can, however, be circumvented by removing the water from the column effluent prior to its evaporation by the interface. Kalasinsky et al.16,17 used the reaction of 2,2-dimethoxypropane with water for (5) Messerschmidt, R. G., Harthcock, M. A, Eds. IR Microspectroscopy: Theory and Applications; Marcel Dekker: New York, 1988; Chapter 14. (6) Lange, A. J.; Griffiths, P. R.; Fraser, D. J. J. Anal. Chem. 1991, 63, 782. (7) Lange, A. J.; Griffiths, P. R. Appl. Spectrosc. 1993, 47, 403. (8) Raynor, M. W.; Bartle, K. D.; Cook, B. W. J. High Resolut. Chromatogr. 1992, 15, 361. (9) Somsen, G. W.; van de Nesse, R. J.; Gooijer, C.; Brinkman, U. A. Th.; Velthorst, N. H.; Visser, T.; Kootstra, P. R.; de Jong, A. P. J. M. J. Chromatogr. 1991, 552, 635. (10) Somsen, G. W.; van Stee, L. P. P.; Gooijer, C.; Brinkman, U. A. Th.; Velthorst, N. H.; Visser, T. Anal. Chim. Acta 1994, 290, 269. (11) Robertson, R. M.; de Haseth, J. A.; Kirk, J. D.; Browner, R. F. Appl. Spectrosc. 1988, 42, 1365. (12) Robertson, R. M.; de Haseth, J. A.; Browner, R. F. Appl. Spectrosc. 1990, 44, 8. (13) Turula, V. E.; de Haseth, J. A. Appl. Spectrosc. 1994, 48, 1255. (14) Robertson, A. M.; Wylie, L.; Littlejohn, D.; Watling, R. J.; Dowle, C. J. Anal. Proc. 1991, 28, 8. (15) Robertson, A. M.; Littlejohn, D.; Brown, M.; Dowle, C. J. J. Chromatogr. 1991, 588, 15. 0003-2700/96/0368-0746$12.00/0
© 1996 American Chemical Society
Figure 1. Schematic of the chromatographic, on-line extraction and interface setup.
postcolumn conversion of the eluent water to the volatile methanol and acetone. Conroy and co-workers18 proposed on-line extraction of the RP-LC effluent with dichloromethane. After separation of the two phases, the organic phase was deposited onto potassium chloride, and spectra were measured by diffuse reflection infrared detection (DRIFT). This study clearly demonstrated the feasibility of postcolumn on-line extraction for RP-LC-FT-IR, but the analytical system used was mechanically rather complex. It did not allow the use of acetonitrile-water eluents, and no experimental data on the use of eluent buffer salts were reported. Furthermore, the DRIFT performance is easily affected by small disturbances of the powdered substrate. Taylor and co-workers19,20 used the postcolumn on-line extraction concept in conjunction with a conventional LC-FT-IR flow cell interface, but still serious spectral interferences due to the organic solvent and coextracted methanol and water were observed. In three previous papers, we showed that RP-LC and FT-IR can be coupled efficiently using a spray-jet assembly interface.9,10,21 The usefulness of the LC-FT-IR system in the impurity profiling of drugs21 and in the identification of isomers in complex mixtures10 was demonstrated. This system, however, was limited with regard to chromatographic flow rate (20-30 µL/min), water content of the eluent (up to 20%), and use of buffers. In the present study, an on-line liquid-liquid extraction module (LLE), developed in our laboratory,22 was inserted between the LC column outlet and the spray-jet assembly interface. The resulting LC-LLE-FT-IR system allows the use of buffered eluents with high water percentages at flow rates up to 0.2 mL/min and yields improved concentration detection limits. EXPERIMENTAL SECTION A schematic of the LC setup with the postcolumn extraction system and the interface is shown in Figure 1. Chromatography. A Gilson (Villiers-le-Bel, France) 302 pump equipped with a Gilson 802c manometric module and a homemade pulse damper was used with a Valco (Houston, TX) six(16) Kalasinsky, K. S.; Smith, J. A. S.; Kalasinsky, V. F. Anal. Chem. 1985, 57, 1969. (17) Kalasinsky, V. F.; Whithead, K. G.; Kenton, R. C.; Smith, J. A. S.; Kalasinsky, K. S. J. Chromatogr. Sci. 1988, 25, 273. (18) Conroy, C. M.; Griffiths, P. R.; Duff, P. J.; Azarraga, L. V. Anal. Chem. 1984, 56, 2636. (19) Johnson, C. C.; Hellgeth, J. W.; Taylor, L. T. Anal. Chem. 1985, 57, 610. (20) Hellgeth, J. W.; Taylor, L. T. Anal. Chem. 1987, 59, 295. (21) Somsen, G. W.; Gooijer, C.; Brinkman, U. A. Th.; Velthorst, N. H.; Visser, T. Appl. Spectrosc. 1992, 46, 1514. (22) de Ruiter, C.; Wolf, J. H.; Brinkman, U. A. Th.; Frei, R. W. Anal. Chim. Acta 1987, 192, 267.
port valve equipped with a loop of 10 or 150 µL. Separations were carried out on a 200 mm × 2.1 mm i.d. column packed with 5 µm Rosil C18 (Research Separations Laboratories, Eke, Belgium); two columns (I and II) of this type were used. Acetonitrile-0.01 M potassium phosphate buffer (pH 7) (40:60 v/v) was used as eluent at a flow rate of 0.2 mL/min. On-Line Extraction and Phase Separation. The organic phase (dichloromethane) was delivered by an Applied Biosystems (Foster City, CA) 400 solvent delivery system at a flow rate of 0.2 mL/min. A home-made pulse damper and a 200 mm × 2.1 mm i.d. column packed with 5 µm material were used to obtain optimal pulse damping. The organic phase was added to the aqueous LC effluent via a 0.25 mm i.d. T-piece. The resulting segmented stream was directed to a 1.5 m × 1.1 mm i.d. stainless-steel coil (helix diameter, 50 mm), which facilitated the extraction. After extraction, the aqueous and organic phases were continuously separated using a home-made sandwich phase separator, which is described in detail in ref 22. With this wetting-type separator, it is possible to obtain a water-free organic phase. The separator has a groove volume of 8 µL and is particularly suitable for aqueous and organic flow rates of ∼0.2 mL/min.22 Stainless-steel restriction capillaries (i.d., 0.12-0.25 mm; length, 100-300 mm) connected to the aqueous separator outlet (waste) were used to regulate the back-pressure; in this way, the amount of organic phase led to the evaporation interface could be varied. The organic outlet flow rate was determined indirectly by measuring the time needed to collect 2.0 mL of the waste flow. For on-line monitoring, a Kratos Analytical (Ramsey, NJ) Spectroflow 757 absorbance detector was used. Interface. After phase separation, the organic stream was led to the spray-jet assembly interface9 via the absorbance detector and a 300 mm × 0.23 mm i.d. fused-silica capillary, which was connected to a stainless-steel needle. A heated nitrogen flow around the needle, which protruded through a nozzle, caused solvent evaporation and ensured deposition of the extracted compounds onto a zinc selenide (ZnSe) window, which was moved by a modified Camag (Muttenz, Switzerland) Linomat III translation table. A ventilation duct connected to a fume hood was placed above the interface; it swept away the generated solvent vapor and kept the dichloromethane concentration in the laboratory air at an acceptably low level. In this study, the interface conditions typically were as follows: needle-protrusion distance, 0.8 mm; needle-to-substrate distance, 0.8 mm; nozzle i.d., 0.60 mm; nitrogen gas pressure, 8 bar; nitrogen gas temperature, 150 °C; table speed, 1.6 mm/min. After deposition, the ZnSe substrate holding the Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
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immobilized analytes was transferred to the FT-IR microscope beam area for the recording of IR spectra. FT-IR. FT-IR transmission measurements were carried out on a Bruker (Karlsruhe, Germany) IFS-85 FT-IR spectrometer equipped with a Bruker A590 FT-IR microscope containing a narrow-range mercury-cadmium-telluride (MCT) detector. A Bruker microscope X-Y stepper table was used to position the ZnSe window in the IR beam. The adjustable microscope aperture typically was square, with the side in the 100-160 µm range. Spectra were recorded in the absorbance mode at 2 cm-1 optical resolution and were baseline corrected. Library searches were performed using the search algorithm of the Bruker ATS89B software. The spectra obtained for the phenylurea herbicides were searched against a library consisting of about 400 pesticide spectra (KBr disk at 2 cm-1 optical resolution). Materials. Demineralized water was purified with a Milli Q system (Millipore, Bedford, MA). Acetonitrile (LC-quality) was obtained from Baker (Deventer, Netherlands) or Biosolve (Barneveld, Netherlands) and methanol (LC-quality) from Rathburn (Walkerburn, UK). Dichloromethane, potassium hydrogen phosphates, and 8-hydroxyquinoline (all analytical-grade) were purchased from Baker and the phenylurea herbicides (96-99% purity) from Riedel-de-Hae¨n (Seelze, Germany). Acenaphthenequinone (tech grade), phenanthrenequinone (95%), and 2,7-dihydroxynaphthalene (99%) were purchased from Aldrich (Milwaukee, WI) and catechol, resorcinol, pyrogallol, 4′-hydroxyacetanilide, benzoin, and 2-hydroxynaphthalene (all analytical-grade) from Merck (Amsterdam, Netherlands). The analytical-grade chemicals estriol, ethynylestradiol, and 4′-ethoxyacetanilide were obtained from Sigma (St. Louis, MO), phenol and caffeine from Janssen Chimica (Geel, Belgium), indole from BDH (Poole, UK), and desmethyldiazepam from Bufa Chemie (Castricum, Netherlands). A 60 mm × 30 mm × 1.5 mm ZnSe window (Biorad, Du¨sseldorf, Germany) was used as deposition medium. Two analyte mixtures were prepared in eluent: test mixture A contained 35-50 µg/mL each of acenaphthenequinone, phenanthrenequinone, diuron, and linuron, and test mixture B contained 30-60 µg/mL each of fenuron, monuron, chlortoluron, diuron, metobromuron, linuron, and chlorbromuron.
RESULTS AND DISCUSSION In the present paper, the potential of on-line extraction-based LC-FT-IR was investigated. First the performance of the extraction module-interface system was evaluated by studying parameters such as extracolumn band broadening, phase separation, interface performance, analyte extraction yield, and use of buffer salts. Subsequently, the total LC-LLE-FT-IR system was considered with emphasis on the quality of the spectral data. Finally, the mass sensitivity of the system was determined, and a method was designed to improve concentration detection limits. Postcolumn On-Line Extraction. In a postcolumn liquidliquid extraction setup, continuous extraction is performed by introducing an immiscible organic solvent to the aqueous LC effluent, thus producing a segmented stream. After equilibrium has been achieved in the extraction coil, the two liquid phases are separated in a phase separator. In the present study, a sandwich-type phase separator was used, which operates on the basis of wetting. After separation, the aqueous phase was sent 748 Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
Figure 2. (A) LC-UV and (B) LC-LLE-UV chromatograms of test mixture A. Injection volume, 10 µL; LC column I; UV detection at 250 nm; SE, 0.89. Further conditions, see Experimental Section. Peaks: AQ, acenaphthenequinone; Di, diuron; PQ, phenanthrenequinone; Li, linuron. Peaks in chromatograms A and B are aligned; in B the extraction causes an overall shift of 3.5 min.
to waste, and the organic phase was led to the solvent elimination interface via a UV absorbance detector (Figure 1). The organic flow through the detector, and thus the phase separation efficiency (SE), could be adjusted by applying backpressure on the aqueous outlet of the phase separator using restriction capillaries. The SE is calculated as the ratio of the organic flow through the detector to the initial organic phase flow. If the organic extractant is able to dissolve (part of) the organic modifier present in the aqueous eluent, SE values above 1.0 can be obtained. However, to ensure a water-free organic phase, in general, the SE has to be set below 1.0; this means that part of the organic solvent is sent to waste. Dichloromethane (DCM) was used as organic phase for basically two reasons. First, DCM is a relatively strong extractant that can extract fairly polar compounds. Second, DCM is highly volatile (bp 40 °C) and can therefore be evaporated efficiently. In the preliminary stage of the study, it was found that the spray-jet assembly interface can handle a 0.2 mL/min flow of pure DCM. Since the sandwich phase separator shows optimum performance at a phase ratio of about 1.0, the eluent flow rate was set to 0.2 mL/min as well, thereby allowing the use of 2.1 mm i.d. LC columns. It is important that the extraction step does not seriously affect the obtained LC separation, i.e., that chromatographic resolution is maintained. As a typical example, Figure 2 shows the LC-UV analysis of test mixture A both with and without postcolumn extraction. It is evident that the extraction system shows proper performance and that the extra band broadening caused by the
extraction is small. Upon extraction, an overall peak height decrease of ∼30% is observed. This decrease is not caused by incomplete extraction (the extraction efficiencies of the analytes are all above 90%; see below) but is mainly due to the fact that the acetonitrile present in the LC eluent dissolves in DCM. Consequently, the total organic phase volume increases, and the concentrations of the extracted analytes decrease correspondingly. Using the principle of summation of variances, the band broadening due to the introduction of the extraction module (σextr) can be estimated from the standard deviation of the on-line recorded peaks obtained with (σtot) and without extraction (σdir). For instance, for the diuron peak in Figure 2, σextr equals 2.1 s, which corresponds with an increase of the peak width at halfmaximum (fwhm) of ∼4%. Since σextr is known to depend on SE,22,23 σextr was determined for diuron at several SE values (range tested, 0.45-1.1) using acetonitrile-0.01 M potassium phosphate buffer (pH 7) (40:60 v/v) as eluent and a flow rate of 0.2 mL/min for both the aqueous and the organic phases. These experiments revealed that σextr decreases with increasing SE; upon going from SE ) 0.45 to 1.1, σextr decreased from 5.2 to 1.6 s. In the LCLLE-FT-IR experiments described below, the adjusted SE was always taken in the 0.8-1.0 interval, which implied that the fwhm increase caused by postcolumn extraction never exceeded 6%. Previous studies involving the spray-jet assembly interface9,10 showed that deposited peaks were broadened by 20-60% compared with on-line detected peaks. This means that in the current setup, the relative contribution of the extraction module to the final fwhm can be neglected. In other words, the deposition interface remains the main source of extracolumn band broadening. Phase Separation Efficiency and Evaporation Efficiency. The experimental setup was tested with respect to phase separation and evaporation efficiency using several eluents varying in type (acetonitrile or methanol) and percentage of organic modifier. For each eluent, the maximum separation efficiency (MSE) and maximum evaporation efficiency (MEE) were determined. The MSE is the highest SE with which no leakage of aqueous phase segments to the organic outlet of the phase separator is observed. The MEE is the highest SE at which the water-free organic outlet flow still can be evaporated completely by the interface under the interface conditions listed in the Experimental Section. The evaporation was checked visually; incomplete evaporation is indicated by small, fast-spreading droplets on the ZnSe surface. The experimental results for the acetonitrile eluents are depicted in Figure 3. For all acetonitrile eluents tested, two immiscible phases were obtained upon on-line extraction with DCM. They could be separated on-line and allowed the collection of a water-free organic phase. The high MSE values (Figure 3) reflect the dissolution of acetonitrile in DCM during extraction. The volume of the organic phase accordingly increases, and the flow at the organic outlet of the phase separator can exceed 0.2 mL/min. At higher acetonitrile contents, more acetonitrile will dissolve, and SE will increase. However, in practice, SE for acetonitrile-containing eluents is limited by the organic flow that can be handled by the interface (Figure 3). MEE values for this type of eluents are in the 0.8-1.0 range, which corresponds with a flow to the interface of 0.16-0.2 mL/min. Since acetonitrile is less volatile than DCM, (23) Maris, F. A.; Nijenhuis, M.; Frei, R. W.; de Jong, G. J.; Brinkman, U. A. Th. Chromatographia 1986, 22, 235.
Figure 3. Dependence of MSE (O) and MEE (b) on the acetontrile content of the aqueous phase. Aqueous phase, acetonitrile or methanol-0.01 M phosphate buffer (pH 7); organic phase, DCM; phase flow rates, both 0.2 mL/min.
the evaporation efficiency is slightly decreased when the organic phase contains more acetonitrile. This is observed as a small decrease of MEE with an increasing acetonitrile content of the eluent. For eluents containing 0-60 vol % methanol, a constant MSE value of about 0.9 was found. Evidently, DCM does not extract significant amounts of methanol. In other words, for these eluents, the final organic phase essentially consists of pure DCM, which could be handled efficiently by the interface. This means that for methanol eluents, MEE equals MSE. When using methanol contents higher than 60 vol %, the aqueous and organic phases become completely miscible, and phase separation cannot be achieved. Two conclusions can be drawn: (i) the on-line extraction LCFT-IR setup allows the use of both acetonitrile and methanol (up to 80 and 60 vol %, respectively) and (ii) SE should be adjusted to ∼0.9 to ensure both a good interface performance and a favorable IR sensitivity. Extraction Efficiency. To ensure a broad application range of the on-line extraction system, the organic phase should be able to extract a fairly large number of compound classes out of an aqueous eluent. That is, chlorinated solvents such as chloroform, DCM, and dichloroethane should be used rather than alkanes like hexane or isooctane. To explore the extraction limits of the present setup in terms of analyte polarity, the extraction percentages (%E) of a number of analytes were determined. The experiments were mainly focused on the effect of the presence of hydroxyl groups on %E. %E was measured using a flow injection (FIA; no LC column) setup with UV detection. The aqueous phase was acetonitrile-0.01 M potassium phosphate buffer (pH 7) (40: 60 v/v) (0.2 mL/min), and the organic phase was DCM (0.2 mL/ min). %E was calculated as the ratio of the analyte peak height measured in the organic phase after on-line extraction (Hextr) and the peak height obtained without extraction (Hdir). Since Hextr is not a result of phase distribution only, Hextr was properly corrected for analyte-independent parameters, such as the adjusted SE and the degree of dilution of DCM by dissolved acetonitrile. This was done by measuring Hdir and Hextr of a compound with known extraction behavior. High extraction yields were found for compounds such as phenylureas, quinones, desmethyldiazepam, caffeine, and indole, indicating that the current postcolumn extraction setup can be used for the effective extraction of medium-polar compounds. Even analytes containing hydroxyl groups, like benzoin and 8-hydroxylAnalytical Chemistry, Vol. 68, No. 5, March 1, 1996
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Figure 4. FT-IR spectra of peaks recorded in LC-LLE-FT-IR of test mixture A. Spectra A-D correspond with LC peaks of AQ, Di, PQ, and Li of Figure 2B, respectively. Number of scans per spectrum, 512. Further conditions, see Figure 2. Since the 3500-2000 cm-1 region of the quinone spectra is rather featureless, only the 2000-700 cm-1 region of these spectra is shown.
quinoline, showed favorable extraction efficiencies (above 90%). However, in some cases, the presence of a hydroxyl moiety in the analyte molecule has a dramatic effect on the extraction yield. This is indicated by the difference in %E for 4′-ethoxyacetanilde (97%) and 4′-hydroxyacetanilide (28%): replacing an ethoxy group by a hydroxyl group causes a 70% decrease of extraction efficiency. The influence of (the degree of) hydroxyl substitution on %E is nicely illustrated by comparing the %E values of a series of hydroxybenzenes, viz., phenol (98%), catechol (63%), resorcinol (50%), hydroquinone (24%), and pyrogallol (2%). With monohydroxy substitution, quantitative extraction is obtained, whereas only moderate extraction is achieved for the dihydroxybenzenes, with the substitution pattern playing a distinct role. Pyrogallol, which contains three hydroxyl groups, evidently is too polar to show significant extraction. Finally, the fairly good extraction yields found for 2,7-dihydroxynaphtahalene (92%), ethynylestradiol (97%), and estriol (89%) indicate that the effect of the hydroxyl substitution is readily compensated by increasing the apolar moiety of the analytes. The influence of the acetonitrile content of the aqueous phase on %E was also examined briefly. In general, for polar compounds, a small decrease in extraction yield was observed on decreasing the acetonitrile percentage. This effect may be ascribed to changes in the amount of acetonitrile transferred from the eluent to DCM. The above extraction experiments indicate that, with the present system, a wide range of compounds can be efficiently extracted on-line. In other words, with this approach, the applicability range of our LC-FT-IR setup can be increased significantly compared with direct eluent elimination.9,10,21 In the latter situation, the restricted water content of the eluent (max 20%) seriously limits the number of compounds that can be analyzed by RP-LC-FT-IR. Use of Buffer. An interesting feature of the present system is that buffered eluents can be used. RP-LC eluents frequently contain nonvolatile buffer salts that cannot be eliminated by an 750 Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
evaporation interface. When such an eluent is led directly to the spray-jet assembly, the salt(s) will be deposited together with the analytes, causing unwanted spectral interferences. Moreover, interface clogging and disturbance of the analyte deposition process may occur. The use of volatile buffers can eliminate most of these problems. Still, particularly at low analyte levels, absorption bands due to the buffer are often observed,7,12 and spectral subtraction will be needed. Since buffer salts are not soluble in DCM, they are not extracted during postcolumn extraction, and consequently they do not interfere. In fact, throughout this study, the aqueous LC eluent was buffered with potassium hydrogen phosphates (0.01 M), and no interferences with regard to phase separation, deposition, or IR measurement were observed. LC-LLE-FT-IR. To perform LC-LLE-FT-IR analysis, the organic outlet of the phase separator was connected to the sprayjet assembly interface. The optimum needle-protrusion distance and the needle-to-substrate distance were both found to be 0.8 mm, which is similar to the conditions used in previous studies.9,10,21 However, to ensure complete evaporation of the organic phase, the nitrogen gas pressure had to be raised to 8 bar and the temperature to 150 °C. No boiling of the organic phase was observed under these conditions, which indicates that the solvent evaporation causes a considerable cooling of the needle tip. To examine the total LC-LLE-FT-IR system, the two test mixtures A and B were analyzed. The phenylureas were selected to establish whether the present system can be used to analyze (thermolabile) polar pesticides. The on-line-extracted LC chromatogram of test mixture A (Figure 2B) was immobilized on ZnSe. The width of the deposited spots was ∼0.2 mm. Upon visual inspection by microscope, the quinone spots appeared like a well-defined smooth film of very small crystals. The two phenylureas, however, yielded discontinuous spots showing many small, irregular domains. Nevertheless, good-quality spectra (Figure 4) were obtained from every LC peak by FT-IR microscopic measurement of the centers of the deposited
spots. The band positions and intensities of the spectra nicely matched those of the corresponding KBr reference spectra, as was verified by spectral library search. Next, test mixture B (10 µL injection) was analyzed by LCLLE-FT-IR, testing the capability of the system to distinguish between structurally similar compounds. The seven phenylureas were baseline separated (not shown), on-line extracted (SE, 0.86), and deposited onto ZnSe. Good IR spectra with a quality similar to those of Figure 4, spectra B and D, were obtained for all injected compounds. The recorded herbicide spectra are, of course, rather similar, but the spectral differences in the fingerprint region (1500-700 cm-1) allowed the correct and unambiguous library identification in all but one case. The spectrum of the LC peak at tR ) 20 min was attributed to an unspecified herbicide residue (sample code PT11B; hit quality (Hq), 389), while the target compound (metobromuron) came out second best, with an Hq of 373. The latter result deserves some attention. From the search results, it was suspected that none of the library reference spectra adequately matched the recorded spectrum. This could be confirmed by comparing the library spectrum of metobromuron with the spectrum obtained after LC-LLE-FT-IR. The library spectrum shows sharp bands in the fingerprint region; the bands in the recorded spectrum are somewhat broader and less pronounced. In a previous study,21 similar spectral differences were attributed to morphological differences. In the present study, the metobromuron reference spectrum originated from a crystalline sample; the LC-LLE-FT-IR spectrum of metobromuron, however, was recorded from a fluidlike deposit. We succeeded in obtaining the appropriate (“amorphous”) metobromuron reference spectrum, by air-drying a drop of a 1 mg/mL solution of metobromuron in acetonitrile-water (80:20 v/v) on a ZnSe window. The recorded FT-IR spectrum of a noncrystalline region of the dry spot showed broadened bands; the spectrum was added to the spectral library. A subsequent library search of the LCLLE-FT-IR spectrum of metobromuron gave an Hq of 769 for amorphous metobromuron, with PT11B (Hq, 389) and crystalline metobromuron (Hq, 373) in second and third place, respectively. The spectra of Figure 4 indicate that the limit of identification of the present LC-LLE-FT-IR method (using 10 µL injections) is ∼3 µg/mL. This corresponds to a minimum identifiable quantity (MIQ) of ∼30 ng injected. Because acetonitrile dissolves in DCM, the organic phase volume increases during extraction. This means that using an eluent containing 40 vol % acetonitrile and an adjusted SE of 0.9, 36% of the resulting organic phase goes to waste. In other words, when 30 ng of analyte is injected and quantitatively extracted, about 20 ng will actually reach the interface and be analyzed by FT-IR. This estimate is in good agreement with previously reported LC-FT-IR identification limits using the same interface for direct deposition.9,10 Improvement of Concentration Detection Limits. The minimum identifiable concentration (MIC) of the analytes of interest can be decreased considerably by enlarging the injection volume. Large volume injections without serious loss in separation efficiency can be accomplished by dissolving the sample in a solvent that has a lower elution strength than the eluent. The analytes will then be trapped at the top of the column (on-column focusing), and no undue band broadening will occur.
Figure 5. (A) LC-LLE-UV chromatogram of dilute test mixture B. Analyte concentrations, 2-4 µg/mL; injection volume, 150 µL; LC column II; UV detection at 250 nm; SE, 0.9. Further conditions, see Experimental Section. Peaks: Fe, fenuron; Mo, monuron; CT, chlortoluron; Di, diuron; MB, metobromuron; Li, linuron; CB, chlorbromuron. Baseline disturbance at t ) 6-8 min is caused by the injected solvent plug. (B) FT-IR spectrum of peak CT recorded in LCLLE-FT-IR of dilute test mixture B. Number of scans, 128.
Figure 6. FT-IR spectrum of acenaphthenequinone recorded in LC-LLE-FT-IR after injection of 150 µL of a 180 ng/mL acenaphthenequinone solution in buffer on LC column II. Number of scans, 1000. Further conditions, see Experimental Section.
Using a dilution of test mixture B in buffer (2-4 µg/mL in 3 vol % acetonitrile in buffer), it was experimentally assessed that, in the present system, the injection volume can be increased from 10 to 150 µL without impairing the chromatographic resolution. Also, phase separation appeared to be undisturbed by the injection of a large volume of a solution differing in composition from the eluent. Figure 5A shows the on-line-extracted chromatogram (UV detection) of a 150 µL injection of the diluted phenylurea mixture. It is clear that the baseline separation is fully maintained. The chromatogram was deposited on ZnSe, and good-quality spectra of all analytes were recorded. As an example, the spectrum of chlortoluron is shown in Figure 5B. All spectra were unambiguously library identified; the spectrum of the metobromuron peak was (correctly) attributed to amorphous metobromuron by the library search program. To determine MIC values, various concentrations of acenaphthenequinone (in buffer) were injected (150 µL) and deposited. Using 1000 scans, the deposit from a 90 ng/mL injection could be detected. To obtain an identifiable spectrum, 180 ng/mL had to be injected (Figure 6). This Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
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concentration sensitivity represents a considerable improvement over previous reported RP-LC-FT-IR methods, which yield MICs in the (high) micrograms per milliliter range.3-20 In summary, postcolumn extraction in combination with solvent elimination considerably enlarges the applicability of RP-LC-FTIR. On-line extraction of the aqueous LC eluent with a volatile organic solvent circumvents inherent limitations of direct eluent evaporation. Eluents containing nonvolatile buffer and with a high water percentage can be handled at a flow rate as high as 0.2 mL/min, enabling use of conventional LC equipment. The LCLLE-FT-IR system yields identifiable IR spectra, which are searchable against KBr reference spectra.
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ACKNOWLEDGMENT The present study was supported by the Dutch Foundation of Technical Sciences (STW) under Grants Nos. 700-349-3024 and 790-600-3024.
Received for review July 10, 1995. Revised manuscript received December 1, 1995. Accepted December 4, 1995.X AC950686R X
Abstract published in Advance ACS Abstracts, January 15, 1996.
